This invention relates to spectrophotometry and the spectrophotometric analysis of blood samples. In particular, this invention relates to a method and apparatus for providing a non-destructive pre-test screen of specimen integrity for a blood analyzer by measurement of absorbance or reflectance.
Clinical laboratory tests are routinely performed on the serum or plasma of whole blood. In a routine assay, red blood cells are separated from plasma by centrifugation, or red blood cells and various plasma proteins are separated from serum by clotting prior to centrifugation.
Haemoglobin (Hb), bilirubin (Bili) and light-scattering substances like lipid particles are typical substances which will interfere with, and affect spectrophotometric and other blood analytical measurements. Such substances are referred to as interferents. Elevated Bili can be due to disease states, and increased lipid particles in the blood, also known as hyperlipidemia, can be due to disease states and dietary conditions. Elevated Hb in the blood, hemoglobinemia, can be due to disease states and as a result of specimen handling. Clinical laboratories currently emphasize these three potential interferents as being of greatest concern with respect to affecting blood analysis. Biliverdin, (BV), a fourth potential interferent, is rarely mentioned.
BV is the precursor of Bili, and if Bili becomes oxidized, it will revert to BV. BV and Bili are two of a class of compounds referred to as bile pigments. BV is not normally present in serum, but regularly accompanies Bili in the serum of patients with carcinomatous obstruction of the bile duct, and is frequently found in the blood of patients with liver cirrhosis, and bile duct occlusion by gallstones.
Upon visual inspection, Bili gives a yellow or orange colour to serum and 10 plasma and is considered to be the major bile pigment in serum or plasma. Bili is sometimes described as a greenish/yellow pigment (Guyton, A. C. and Hall, J. E., Textbook of Medical Physiology, 1996, page 886). However, it is likely BV, rather than Bili, that is responsible for the greenish colour. In fact, these specimens may, nevertheless, have acceptable levels of Bili. A yellow colour chart is available for visual grading of Bili levels in serum or plasma, but there is no known colour chart for green bile pigment or BV. Visual inspection may still provide any opportunity for rejection of green specimens, however, for automated systems, no method exists for screening serum or plasma specimens for increased blood levels of BV, also known as hyperbiliverdinemia.
Many tests conducted on plasma or serum samples employ a series of reactions which terminate after the generation of chromophores which facilitate detection by spectrophotometric measurements at one or two wavelengths. Measurement of interfering substances prior to conducting such tests is important in providing meaningful and accurate test results. In fact if a sample is sufficiently contaminated with interferents, tests are normally not conducted as the results will not be reliable.
Specimen integrity is an essential part of quality assurance as it directly affects the accuracy of test results. The presence of interferents in a plasma or serum sample compromises specimen integrity.
Spectrophotometric measurement uses infrared (IR) or near infrared radiation (NIR) to assess the concentration of various constituents in a blood sample. Examples of photometric measurements using containers which hold a blood sample are disclosed in U.S. Pat. Nos. 5,291,884; 5,288,646; 5,066,859; and 5,366,903.
U.S. Pat. No. 5,366,903 discloses a sampling device which allows photometric quantitative determination of an analyte in whole blood. The device overcomes the problems of having blood cells in a blood sample by effectively xe2x80x9csqueezing outxe2x80x9d red blood cells and providing a small volume of sample, free of red blood cell material, from which particular analytes can be measured.
Other applications of photometric methodology include non-invasive determinations of analyte concentrations such as described in U.S. Pat. Nos. 5,360,004; 5,353,790; and 5,351,685. However none of these documents discloses a method of measuring interferents in the plasma or serum of a blood sample, in order to assess specimen integrity for further analysis.
Current methods used for detecting haemoglobinemia, bilirubinemia and lipemia or turbidity utilize visual inspection of the specimen with or without comparison to a coloured chart. It is to be understood that those practising in the field use the terms lipemia and turbidity interchangeably. This is so because lipemia is the major cause of turbidity in serum or plasma.
Visual inspection is sometimes employed on a retrospective basis where there is a disagreement between test results and clinical status of the patient in order to help explain such discrepancies.
A sample of plasma or serum is normally transferred from an original or primary tube or container to a secondary tube or container. These secondary tubes are amber coloured to protect photo sensitive constituents. The amber colouring makes visual inspection virtually impossible. On occasion, labels cover portions of the tube further restricting a full visual examination.
Pre-test screening of specimens by visual inspection is semi-quantitative at best, and highly subjective and may not provide sufficient quality assurance as required for some tests.
Furthermore, visual inspection of specimens is a time consuming, rate limiting process. Consequently, state-of-the-art blood analyzers in fully and semi-automated laboratories do not employ visual inspection of specimens.
Other methods to assess specimen integrity employ direct spectrophotometric measurement of a diluted sample in a special cuvette. However, such methods are not rapid enough for screening samples. In order to obtain a measurement of the sample of the plasma or serum, primary specimen tubes, or containers, must be uncapped, a direct sample of the specimen taken and diluted prior to measurement. Both of these steps are time consuming and require specialized and/or disposable cuvettes.
It is desirable to provide an apparatus and a method whereby specimen integrity is rapidly and accurately assessed without disturbing the sample. The disadvantages of the prior art may be overcome by providing a rapid and accurate method and apparatus for monitoring blood specimen integrity before samples are presented for analysis.
In one aspect of the invention, spectral data is used in a novel way so as to determine if the specimen which is presented for such analysis in a primary container contains interferents and if so, to what extent.
In another aspect of the invention, there is provided an apparatus and a method for determining blood specimen integrity of a specimen contained in a primary container using a spectrophotometer to irradiate and measure radiation from the specimen.
In a further aspect of the invention, there is provided an apparatus and a method for determining blood specimen integrity, where a primary sample tube containing a specimen has a sample identification label on the exterior surface of the sample tube, by using a spectrophotometer to irradiate and measure radiation from the specimen.
In yet another aspect of the invention there is provided an apparatus and a method for determining blood specimen integrity of a specimen contained in a primary container where light is transmitted through the label, container and specimen.
In yet a further aspect of the invention, there is provided an apparatus and a method for determining blood specimen integrity of a specimen contained in a primary container, where the light is reflected from the backside of a label after penetrating the specimen and container through an unlabelled section of the container.
In another aspect of the invention, specimen integrity of a specimen contained in a primary container is assessed by measuring:
1. Haemoglobin concentration as an assessment of haemolysis;
2. Bilirubin concentration as an assessment of bilirubinemia;
3. Biliverdin concentration as an assessment of biliverdinemia; and
4. Equivalent intralipid concentration for the assessment of turbidity.
Bile pigments may be measured in accordance with the method and apparatus of the present invention. According to one aspect of the present invention, Hb concentration is determined by measurement of absorption of different wavelengths of light in serum or plasma specimens which are then compared with values obtained through calibration using reference measurements for haemoglobin in serum or plasma specimens. Bili and/or BV_concentration is determined by measurement of absorption of different wavelengths of light in serum or plasma specimens which are then compared with values obtained through calibration using reference measurements for Bili and/or BV in serum or plasma samples. Turbidity is determined by measurement of absorption of different wavelengths of light in serum or plasma specimens which are then compared with values obtained through calibration using serum samples spiked with known amounts of intralipid (IL). Intralipid is a fat emulsion in water which is similar to naturally-occurring chylomicrons and is used to simulate turbidity. On the basis of the results from measurements of any one or more of these interferents at a time, in comparison with reference measurements of various levels of interferents, a decision is made concerning whether to reject or accept the sample.
According to another aspect of the present invention, Hb concentration is determined by measurement of reflectance of different wavelengths of light in serum or plasma specimens which are then compared with values obtained through calibration using reference measurements for haemoglobin in serum or plasma samples. Turbidity is determined by measurement of reflectance of different wavelengths of light in serum or plasma specimens which are then compared with values obtained through calibration using serum samples spiked with known amounts of intralipid (IL). Bili and/or BV concentration is determined by measurement of reflectance of different wavelengths of light in serum or plasma specimens which are then compared with values obtained through calibration using reference measurements for Bili and/or BV in serum or plasma samples. On the basis of the results from measurement of any one or more of these interferents at a time, _in comparison with reference measurements of various levels of interferents, a decision is made concerning whether to reject or accept the sample.
In its broad aspect the present invention provides an apparatus for determining specimen integrity of a specimen contained in a primary container where the apparatus comprises: a housing for receiving a sample; a radiation source; a sensor; a means for optically connecting the radiation source with the sensor along a sample path through the housing and along a reference path which by-passes the sample; a means for selectively passing a beam from the sample path and from the reference path to the sensor; and a means for correlating a sensor response, from the sample path relative to a sensor response from the reference path, to a quantity of a known substance in said sample. The housing has a cavity for receiving a sample and a lid for selectively opening and closing the cavity. The radiation source is for emitting a beam of radiation, and the sensor is responsive to receipt of radiation.
In a further aspect the present invention provides an apparatus for determining specimen integrity of a of a specimen contained in a primary container where the apparatus comprises a housing which contains a cavity for receiving a sample and a lid for selectively opening and closing said cavity. It is understood that any means for excluding from the sample, light other than that from the radiation source of the apparatus, is within the scope of this invention. Also, if dark current, i.e., sensor response when sensor is not exposed to the instrument light, is subtracted from both the reference and sample measurements, the room light impinging on the detector can be effectively subtracted without affecting the instrument performance significantly.
The apparatus further comprises a quartz-tungsten-halogen bulb capable of emitting a near infrared, and adjacent visible region light beam having wavelengths from 475 nm to 1080 nm and a beam splitter for splitting the light beam from the quartz-tungsten-halogen bulb into a sample path beam for travel along a sample path and a reference path beam for travel along a reference path. This apparatus further comprises a shutter for selectively blocking the sample path light beam which travels along the sample path and the reference path light beam which travels along the reference path, as well as optical fibre bundles for transmitting the sample path light beam through a sample enclosed in the housing, and optical fibre bundles for transmitting the sample path light beam from the sample to a beam combiner. The beam combiner combines the beam from the sample path and the beam from the reference path into a combined beam. The combined beam of this apparatus is then passed onto a sensor. This apparatus further comprises a series of mirrors along the reference path and the sample path to optically connect the near infrared, and adjacent visible region light source with the sensor, and a grating for dispersing the combined beam into component wavelengths which are passed onto the sensor. The sensor of this apparatus is a photodiode array comprised of a plurality of pixels wherein each of the pixels is set to measure one of a plurality of predetermined light frequencies. Based on the measurement of the frequencies, the sensor generates a plurality of signals wherein each of the signals is responsive to an amount of radiation received by each of the pixels. This apparatus further comprises an analog-to-digital converter to generate digital information from the plurality of signals and a microprocessor, which is connected to the convertor, to correlate the digital information to a quantity of a known substance in the sample.
In a further aspect of the present invention the apparatus provides a means for determining specimen integrity of a of a specimen contained in a primary container by determining the concentrations of a known substance which is selected from a group comprising haemoglobin, bilirubin, _biliverdin and intralipid.
In yet a further embodiment of the present invention a method is provided for determining serum and plasma specimen integrity of a specimen contained in a primary container, wherein the method comprises the following steps. First, transmitting a beam of radiation along a sample path through a sample and along a reference path by-passing the sample. Next, selectively receiving the beam of radiation from the sample path and the reference path, and analyzing the received beams of radiation from the sample path and from the reference path for an amplitude of at least one predetermined light frequency. Finally, correlating the amplitude of the at least one predetermined light frequency with a quantity of a known substance.
In a further aspect of the present invention there is provided a method for determining serum and plasma specimen integrity of a specimen contained in a primary container, wherein the method allows for the determination of the concentrations of a known substance which is selected from a group comprising haemoglobin, bilirubin, biliverdin and intralipid.
In a further embodiment of the present invention there is provided a method whereby the method for determining serum and plasma specimen integrity of the present invention is carried out on one apparatus of the present invention and a second apparatus is used, based on the calibrations of the first apparatus to provide accurate determinations of interferents on the second apparatus, without calibration of the second apparatus.
In yet a further embodiment of the present invention there is provided a method whereby specimen integrity may be determined on a variety of primary specimen container types without recalibration of the apparatus in use to determine interferent concentration and type. Such container types include pipette tips, PVC tubing, as well as tubes, cuvettes, syringe barrels all made of plastic or glass materials.
In a further embodiment there is provided a method for measuring the concentration of interferents in specimens contained in different container types using different analyzers without calibrating the subsequent analyzers for container type or the different analyzer.
In still another aspect of the present invention, a method for rejecting a sample contained in a sample container from further clinical assay based on determining the concentration of at least one interferent in the sample, comprises the steps of:
positioning the sample container in a spectrophotometer such that the sample can be irradiated by the spectrophotometer;
irradiating the sample with at least one frequency of radiation;
correlating absorbance of the radiation by the sample with a standard for the interferent(s) to determine the concentration of the interferent(s);
and
rejecting the sample if the concentration of the interferent(s) exceeds a predetermined criteria.
Yet another aspect of the present invention provides a method for rejecting a plasma sample contained in a sample container from further clinical assay based on determining the concentration of at least one interferent in the sample, where the method comprises the steps of:
positioning the sample container in a spectrophotometer such that the plasma sample can be irradiated by the spectrophotometer;
irradiating the plasma sample with at least one frequency of radiation;
correlating absorbance of the radiation by the plasma sample with a standard for the interferent(s) to determine the concentration of the interferent(s) including calculating the first derivatives of at least two portions of a spectrum generated from a scan for a particular interferent which are used in an algorithm in respect of the interferent(s) to calculate the particular interferent(s) concentration(s); and
said algorithm(s) in respect of hemoglobin, bilirubin and intralipids are, respectively:
a. In [(g/L hemoglobin)+1]=7.58(603 nm)+11.75(679 nm) 21.50(1,044 nm)+0.31
where (Xnm) is the first derivative of the value of an absorbance measured at the wavelength specified;
b. xcexcmoles/L bilirubin=xe2x88x923601(641 nm)+3415(662 nm)+12710(731 nm)xe2x88x928214(763 nm)xe2x88x92120
where (Ynm) is the first derivative of the value of an absorbance measured at the wavelength specified; and
c. In (g/L intralipids)=1.53(975 nm)xe2x88x928.48where (Znm) is the raw absorbance measured at the wavelength specified; and
rejecting the plasma sample if the concentration of the interferent(s) exceeds a predetermined criteria.
Another aspect of the present invention features a method for rejecting a sample contained in a sample container from further clinical assay based on determining the concentration of at least one interferent in the sample, where the method comprises the steps of:
positioning the sample container in a spectrophotometer such that the sample can be irradiated by the spectrophotometer;
irradiating the sample with at least one frequency of radiation;
correlating reflectance of the radiation by the sample with a standard for the interferent(s) to determine the concentration of the interferent(s); and
rejecting the sample if the concentration of the interferent(s) exceeds a predetermined criteria.